Reliable and effective control of plant development, including growth and reproduction, continues to be a challenge for plant scientists. One way to accomplish this control is to apply various plant treatment agents that cause plants to exhibit desired characteristics. Unfortunately, this is often only a reliable method when the treatment thoroughly contacts one or more specific tissues that are difficult to reach, such as tissues inside of the plant.
Soaking plants for a prolonged period can deliver some agents to desired tissues. However, this approach often leads to undesired effects, such as increased mortality due to the agent being toxic to the plant in a prolonged or non-specific exposure.
The use of doubled haploids (DH) allows breeders to generate completely homozygous and homogenous lines in fewer generations than traditional backcrossing (Eder and Chalyk, 2002; Röber et al., 2005; Chang and Coe, 2009; Geiger, 2009). DH techniques have been developed for over 250 crop species (Forster and Thomas, 2005) and DH lines have been used for structural and functional genomics, marker-trait association studies, and molecular cytogenetics (Chang and Coe, 2009; Geiger, 2009). Incorporating DH technologies in a plant breeding pipeline can increase efficacy of selection (Röber et al., 2005; Geiger, 2009; Geiger and Gordillo, 2009), reduce breeding cycle length (Szarejko and Forster, 2007; Chang and Coe, 2009; Geiger and Gordillo, 2009), and reduce efforts required for line maintenance (Röber et al., 2005).
Although spontaneous chromosome doubling occurs, the frequency is so low (typically less than 5%), that researchers attempting to create doubled haploids plants (collectively termed DH) often subject haploid plants to a treatment that promotes chromosome doubling. Haploid seedlings subjected to a chromosome doubling treatment (termed DH0 plants) can produce haploid egg and/or sperm, and if the DH0 plants are successfully selfed, the zygotic chromosome number can be recovered in substantially homozygotic offspring (termed DH1 plants) that exhibit the vigor and fertility expected of 2n sporophytes.
A common method of artificially triggering chromosome doubling is to apply the anti-microtubule agent colchicine (Chase, 1952, 1969; Gayen et al., 1994; Bordes et al., 1997; Chalyk, 2000; Eder and Chalyk, 2002; Han et al., 2006). However, this was considered an unreliable approach because the effects were often genotype specific (Geiger, 2009) and the concentrations of colchicine needed to bring about improved doubling rates proved to be toxic to treated seedlings (Jensen, 1974). Today, institutions attempting to provoke chromosome doubling are actively exploring treatments which are less toxic to plant tissue and less dangerous to human technicians conducting the treatments (Geiger and Gordillo, 2009).
Gayen et al. (1994) removed the tips of seedling coleoptiles and subjected the remaining body of the seedlings to an extended (6+ hours) soak in a low colchicine concentration (0.1% or less) to generate a doubling rate of 18.05%. Deimling et al. (1997) improved this method by waiting to remove tips until the coleoptiles were at least 1 cm long and soaking the plants in 0.06% colchicine and DMSO for 12 hours in a dark room. Eder and Chalyk (2002) demonstrated that this procedure works on a range of genotypes, with an average success rate of nearly 50%. However, none of these methods are amenable to the sort of high-throughput processes needed in an industrial setting, nor do they generate the rate of doubling needed to make the practice a highly efficient industrial procedure.